The Escherichia coli catabolite gene activator protein (CAP) is a well characterized sequence-specific DNA binding and DNA bending protein involved in regulation of gene expression. The crystallographic structure of CAP has been determined to 2.5 Angstroms resolution, and the crystallographic structure of the CAP-DNA complex has been determined to 3.0 Angstroms resolution. Our research project regarding DNA binding. DNA bending, and transcription activation by CAP is directed at four long-range objectives: (i)to understand the structural and thermodynamic basis of DNA specificity by CAP, (ii) to understand the structural and thermodynamic basis of CAP-induced DNA bending, (iii) to understand the role of CAP-induced DNA bending in regulation of gene expression, and (iv) to understand the role of protein-protein contact between CAP and RNA polymerase in regulation of gene expression. Specific Aim 1: Analysis of DNA binding by CAP. Experiments are proposed to analyze the effects of substitution of an amino acid of CAP that participates in an amino acid-base pair contact in the CAP-DNA complex. Specific Aim 2: Analysis of DNA bending by CAP. Experiments are proposed to develop a fluorescence energy transfer assay for analysis of protein- induced DNA bending in solution, to determine the DNA bend angle and the DNA trajectory in the CAP-DNA complex in solution, to analyze mutants of CAP defective in DNA bending, to analyze mutants of the DNA site defective in DNA bending, and to determine whether mutants defective in DNA bending are defective in transcription activation. Specific Aim 3: Analysis of the transcription activation surface of CAP. Experiments are proposed to analyze the effects of substitution of an amino acid of CAP thought to participate in a direct CAP-RNA polymerase contact in the CAP-DNA-RNA polymerase ternary complex, and to determine whether transcription activation by CAP involves the activation surface of the promoter-proximal subunit of CAP, the activation surface of the promoter-distal subunit of CAP, or the activation surfaces of both subunits of CAP. Specific Aim 4: Analysis of the transcription activation target of RNA polymerase. Experiments are proposed to isolate and characterize mutants of RNA polymerase alpha subunit specifically defective in response to transcription activation by CAP, to isolate and characterize mutants of RNA polymerase alpha subunit that suppress mutants of CAP specifically defective in transcription activation, to identify the subunit of RNA polymerase closet to the transcription activation surface of CAP in the CAP-DNA-RNA polymerase ternary complex by photocrosslinking, and to identify the amino acid(s) of RNA polymerase closed to the transcription activation surface of CAP in the CAP-DNA-RNA polymerase ternary complex by photocrosslinking. The results to be obtained will be broadly relevant to understanding protein-DNA interaction, protein-induced DNA bending, and regulation of prokaryotic gene expression. Since eukaryotic RNA polymerase subunits and basic transcription factors share sequence and mechanistic homologies with E coli RNA polymerase subunits, the results to be obtained also may be relevant to understanding regulation of eukaryotic gene expression.